Redundancy of Chromatin Remodeling Pathways for the Induction of the Yeast PHO5 Promoter in Vivo

Abstract

Induction of the yeast PHO5 and PHO8 genes leads to a prominent chromatin transition at their promoter regions as a prerequisite for transcription activation. Although induction of PHO8 is strictly dependent on Snf2 and Gcn5, there is no chromatin remodeler identified so far that would be essential for the opening of PHO5 promoter chromatin. Nonetheless, the nonessential but significant involvement of cofactors can be identified if the chromatin opening kinetics are delayed in the respective mutants. Using this approach, we have tested individually all 15 viable Snf2 type ATPase deletion mutants for their effect on PHO5 promoter induction and opening. Only the absence of Snf2 and Ino80 showed a strong delay in chromatin remodeling kinetics. The snf2 ino80 double mutation had a synthetic kinetic effect but eventually still allowed strong PHO5 induction. The same was true for the snf2 gcn5 and ino80 gcn5 double mutants. This strongly suggests a complex network of redundant and mutually independent parallel pathways that lead to the remodeling of the PHO5 promoter. Further, chromatin remodeling kinetics at a transcriptionally inactive TATA box-mutated PHO5 promoter were affected neither under wild type conditions nor in the absence of Snf2 or Gcn5. This demonstrates the complete independence of promoter chromatin opening from downstream PHO5 transcription processes. Finally, the histone variant Htz1 has no prominent role for the kinetics of PHO5 promoter chromatin remodeling.

Isolation of an activator-dependent, promoter-specific chromatin remodeling factor

“…Induction was accomplished by deletion of PHO80, a repressor in the signaling cascade for PHO5 activation (12,26). PHO5 expression, as measured by acid phosphatase activity and mRNA analysis, was only slightly reduced in the absence of Chd1, in agreement with previous reports (13,14), indicating that additional factors may be capable of remodeling PHO5 promoter chromatin in vivo (Fig. 5 A and B).…”

“…Although no single chromatin remodeling complex appears to be essential for activation of PHO5 (13,14), genetic studies have implicated several factors at this locus, including the chromatin remodeling complexes INO80 (15) and SWI/SNF (16), the histone acetyltransferases SAGA (17) and NuA4 (18), and the histone chaperone Asf1 (19,20). The question arises whether the actions of these factors are direct or indirect and whether additional and possibly essential factors could be revealed through unbiased biochemical fractionation of yeast extract.…”

Mediator, TATA-binding Protein, and RNA Polymerase II Contribute to Low Histone Occupancy at Active Gene Promoters in Yeast

“…Yeast harboring the kin28-as analog-sensitive mutation, and the control strain, were grown in CSM-ura containing 1 mg/ml of serine (to allow CHA1 activation) and treated for 1 h with 1 g/ml of NA-PP1 prior to cross-linking. Plasmid pP pho5v33 -lacZ and galactose induction conditions were as reported previously (20,21). RNA Isolation and Northern Analysis-RNA was isolated from yeast cultures grown to an optical density at 600 nm of 0.6 to 1.0.…”

“…Chromatin Immunoprecipitation (ChIP)-ChIP was performed essentially as described previously (3,11,20) with the following modifications: the samples were sonicated using a Diagenode Bioruptor at high power setting, 30-s "on"/30-s "off" for a total of 15 min to obtain sheared DNA 200 -600 bp in length. For induction of the CHA1 gene, 1 mg/ml of serine was added to the medium 30 min before cross-linking.…”

Preferential Repair of DNA Double-strand Break at the Active Gene in Vivo

“…The PHO5 gene is transcriptionally induced in the absence of P i and repressed in the presence of P i (62)(63)(64)(65). Thus, transcription of PHO5 would be repressed in YPD (which contains Pi) and induced in the YPD-P i (YPD without Pi) medium.…”